ABSTRACT Although hypoxia is a major stress on human physiology, several populations have adapted to high altitude (HA) hypoxic environments. Among them, Tibetans have been studied most extensively and they have been shown to have relatively low hemoglobin (Hb) levels. As a result, they are severely hypoxemic and how their physiology copes with hypoxia remains poorly understood. Through a unique combination of genome-wide association studies, tests for polygenic adaptation and functional in vitro assays, the Di Rienzo lab has made two major observations: 1) the strongest association signals for Hb and the strongest selective sweep signals in the genome, found at the EPAS1 locus, influence enhancer activity in endothelial cells (ECs), and 2) lower pulse was favored by natural selection in Tibetans. These findings raise the questions of whether and how cardiovascular functions changed in Tibetans as a result of adaptations to HA hypoxia. To answer them, we propose the following specific aims: Aim 1. Develop a unique resource for the functional characterization of Tibetan adaptations. We will generate and genotype lymphoblastoid cell lines for 24 Tibetans (TBC) and will purchase 24 LCLs from Han Chinese (CHB) from the 1000 genome project. These LCLs will be reprogrammed to induced pluripotent stem cells (iPSCs) and then differentiated in parallel into cardiomyocytes (CMs) and ECs. Aim 2. Test the hypothesis that cardiovascular functions were targets of adaptation to hypoxia in Tibetans. All the CMs and ECs will be cultured in normoxia and hypoxia; RNA-seq data will be collected and analyzed to identify genes and co-expression modules that differ in the transcriptional response to hypoxia between TBC and CHB. Cis-regulatory variants that may account for between-population differences in response to hypoxia will be identified using the RNA-seq data and, in turn, will be tested for evidence of polygenic adaptations in Tibetans. Aim 3. Investigate the effects of adaptive alleles by editing iPSCs. We will use editing technologies to delete 10 enhancers including those at the EPAS1 locus and others identified in Aim 2. We will replace alleles by precise genome editing. We will also generate transgenic mice with deletion of EPAS1 enhancer and with the corresponding enhancer alleles in humans. In summary, we will characterize the mechanisms of cardiovascular adaptations to HA hypoxia through a unique combination of approaches to elucidate the causal basis of a polygenic adaptation to an important environmental stressor, in a non-European population.